Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer

Abstract

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

“…The smaller sizes of the foreign-sequence inserts contained in the segment 7 RNAs of rSA11/NSP3-fNTD, -fRBD, -fExRBD, and -fCR may provide the additional genetic space necessary for re-engineering the S-protein products of these viruses to include routing and localization tags capable of enhancing antigen recognition and processing by immune cells. Particularly valuable may be the inclusion of tags that promote interaction of the S-protein products with antibody heavy-chain (Fc) receptors (e.g., FcRn) [ 50 ], enable aggregation or multivalent presentation of the products [ 51 ], or increase the efficiency of synthesis or secretion of the products [ 52 , 53 , 54 ].…”

Antigenic and immunogenic evaluation of permutations of soluble hepatitis C virus envelope protein E2 and E1 antigens

“…However, it should be noted that sE21E did not elicit higher levels of heterologous NAbs than sE2 and it is possible that sE1 fused directly to the backbone of sE2 negatively impacts folding of sE2 and that incorrectly folded s1E does not. This negative effect of backbone fusing E1 to E2 is supported by a recent study in which the insertion of a cleavable linker sequences between TMtruncated, soluble E1E2 chimeric proteins assists in native protein interaction [57]. It is interesting that sE2 appears to tolerate C-terminal protein fusion better than N-terminal fusion, which is a useful starting point for engineering sE2 protein variants fused to immunostimulatory proteins, such as CR2 receptor-binding domain (p28), as has been used with some success in Dengue virus vaccine research [58].…”

Immunopotentiating and Delivery Systems for HCV Vaccines

“…Some structural alterations in polyphosphazene structure realized in more advanced derivatives, such as poly[di(carboxylatoethylphenoxy)phosphazene], PCEP, which already demonstrated superior in vivo performance with HCV antigens [66]. Due to utmost simplicity of spontaneous self-assembly and co-encapsulation of immunopotentiating molecules, which is achieved by simple mixing of water-soluble components, the system appears to be ideally suited for simultaneous testing of multiple antigens, as well as rapid screening of antigenic constructs designed through computational methods [74].…”